Angiogenic peptides and uses thereof

The present invention relates to peptides that are capable of promoting angiogenesis and to the use thereof in the treatment of angiogenesis-dependent diseases, such as ischemic vascular diseases.

Angiogenesis is the process of generating new capillary blood vessels and involves an interplay between cells and soluble factors (1). In brief, activated endothelial cells migrate and proliferate to form new vessels, which are surrounded by layers of periendothelial cells; small blood vessels are surrounded by pericytes and large blood vessels are surrounded by smooth muscle cells.

Numerous factors regulate the angiogenic process. These include soluble factors and tissue oxygen. In the past two decades, a number of angiogenic molecules which positively regulate the angiogenic process were elucidated. These include Vascular Endothelium Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), acidic FGF/FGF-1, hypoxia-inducible factor-1α (HIF-1α), and others (2). As mentioned, oxygen conditions also have important implications for the physiological and pathological angiogenic process (3). Under hypoxic conditions, VEGF gene expression is induced both in endothelial cells and pericytes to produce secretory forms of VEGF. VEGF, in turn, may bind to VEGF receptor-2 (Kdr) or VEGF receptor-1 (VEGFR-1; Flt-1) expressed on endothelial cells in an autocrine or paracrine manner, thereby causing proliferation of endothelial cells, which may lead to angiogenesis. Basal amounts of vascular VEGF synthesized under normoxia promote the maintenance of microvascular homeostasis (5). Expression of VEGF receptor 1 mRNA (Flt-1) was found to be up-regulated in peri-ischemic endothelial cells and in the infracted core of endothelial cells and periphery, with peak expression of VEGFR-1 in endothelial cells. Gene expression of VEGFR-1 is directly inducible by hypoxia, as in the case of VEGF. Twenty-four hours following hypoxia-induced VEGF gene expression, concurrent with the expression of the VEGFR-1 and 2 (Kdr) genes, endothelial cells begin to proliferate (6, 7).

Hypoxia-inducible gene products that participate in these cellular responses include erytropoietin, VEGF, and glycolytic enzymes (8). Hypoxia can directly enhance the expression of bFGF mRNA in pericytes. Increased expression of bFGF may play an important role in pericyte proliferation and in differentiation of pericytes and smooth muscle cells (9).

Angiogenesis-dependent diseases result when the angiogenic process is disregulated, resulting in excessive amounts of new blood vessels or an insufficient number of blood vessels. Insufficient angiogenesis is related to a large number of diseases and conditions, such as coronary artery diseases and delayed wound healing. To date, cardiovascular diseases are the leading cause of mortality in the United States, Europe, and Israel. In the United States, approximately one million deaths per year are attributed to cardiac causes, fifty percent of which are attributed to Coronary Artery Disease (CAD). The major morbidity from CAD is a result of obstructive coronary artery narrowing and the resultant myocardial ischemia. CAD affects more than 13 million people, and its annual economic burden is in excess of sixty billion U.S. Dollars.

Mechanical revascularization of obstructive coronary stenoses by percutaneous techniques, including percutaneous transluminal angioplasty and stent implantation, is used to restore normal coronary artery blood flow. In addition, coronary artery occlusion bypass surgery is performed using arterial and venous conduits as grafts onto the coronary arterial tree. These treatment modalities have significant limitations in individuals with diffuse atherosclerotic disease or severe small vessel coronary artery disease, in diabetic patients, as well as in individuals who have already undergone surgical or percutaneous procedures.

For these reasons, therapeutic angiogenesis, aimed at stimulating new blood vessel growth, is highly desirable. The therapeutic concept of angiogenesis therapy is based on the premise that the existing potential for vascular growth inherent to vascular tissue can be utilized to promote the development of new blood vessels under the influence of the appropriate angiogenic molecules.

Therapeutic angiogenesis defines the intervention used to treat local hypovascularity by stimulating or inducing neovascularization for the treatment of ischemic vascular disease.

Animal studies have proven the feasibility of enhancing collateral perfusion and function via angiogenic compounds. Those experiments proved that exogenous administration of angiogenic growth factors or their genetic constructs could promote collateral vessel growth in experimental models of chronic ischemia. Although such studies demonstrated proof of concept, additional studies raise issues that still have not been resolved, such as the duration of exposure of the vessels to angiogenic factors and the brief half-lives of such proteins (10).

Synthetic peptides encompassing portions of proteins have become supportive tools for understanding the molecular mechanisms associated with protein biological functions. The use of short peptides constructed from specific regions of human FGF and VEGF that have the potential to efficiently agonize or antagonize the biological functions of the growth factor family members has been described (11). Several groups have reported the use of intact cells to screen a phage display peptide library to identify cell surface-binding peptides (12). A peptide-based ligand receptor map of the VEGF family was constructed by screening human endothelial cells stimulated with VEGF with a peptide library (13). Another study has described the screening of a 12-mer phage display peptide library on VEGF-2 receptor protein (14).

While reducing the present invention to practice, the present inventors used a 12-mer phage display peptide library to uncover peptides which are able to bind the cell-surface of endothelial cells incubated under normoxic or hypoxic conditions. Such peptides were shown to trigger angiogenic processes including endothelial cell-proliferation and vascularization. As such, these peptides can be used to treat various angiogenesis-dependent diseases, such as ischemic vascular diseases. Furthermore, characterization of the nature of endothelial cell signaling by these peptides will provide the basis for the development of targeted angiogenic therapy for morbidities, such as cardiovascular disease.

According to one aspect of the present invention there is provided peptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12.

According to another aspect of the present invention there is provided a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12, the peptide being no more than 50 amino acid residues in length.

According to yet another aspect of the present invention there is provided a peptide comprising an amino acid sequence as set forth in SEQ ID NO:13, 27, or 32, the peptide being at least 6 and no more than 50 amino acid residues in length.

According to still another aspect of the present invention there is provided a composition-of-matter comprising at least two peptides, each independently selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12.

According to an additional aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a peptide having an amino acid sequence as set forth in SEQ ID NO:13, 27, or 32, the peptide being at least 6 and no more than 50 amino acid residues in length, and a pharmaceutically acceptable carrier or diluent.

According to yet an additional aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a peptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12 and a pharmaceutically acceptable carrier or diluent.

According to still an additional aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12, the peptide being no more than 50 amino acid residues in length, and a pharmaceutically acceptable carrier or diluent.

According to a further aspect of the present invention there is provided a method of promoting angiogenesis in a tissue of a subject, the method comprising providing to the subject a therapeutically effective amount of a peptide having an amino acid sequence as set forth in SEQ ID NO:13, 27, 32 the peptide being at least 6 and no more than 50 amino acid residues in length, to thereby promote angiogenesis in the subject.

According to further features in preferred embodiments of the invention described below, the peptide is selected from the group consisting of SEQ ID NOs:2, 6, and 12.

According to still further features in the described preferred embodiments the amino acid sequence is selected from the group consisting of SEQ ID NOs:2, 6, and 12.